During each cell cycle in eukaryotes, the genome must be completely replicated and this replication must begin at the correct time and site (initiation site or origin) (1). Pathogenic viruses often take advantage of this cellular precision to maintain replication of their own genome. The Epstein-Barr virus (EBV) is an orally-transmitted herpesvirus associated with numerous human neoplasms (2) that infects over 90% of the world's population (3). Latent EBV infection stimulates B cell proliferation in vitro, and can lead to B cell transformation (4). Following infection, the EBV genome is maintained in the host cell as a plasmid that is replicated by machinery comprised of several host proteins and a sole EBV protein, the EBV nuclear antigen 1 (EBNA-1). This EBNA-1 protein is required for replication of the EBV DNA genome. EBNA-1 is a DNA-binding protein that binds to an EBV sequence called the viral origin of replication plasmid, OriP (5). Studies demonstrating a role for EBV in Burkitt's lymphoma and Hodgkin's disease (6), multiple sclerosis (7), lupus (8), infectious mononucleosis (9), and nasopharyngeal carcinoma (10), combined with the recent discovery that EBNA-1 induces DNA double strand breaks (11), suggest that inhibitors of EBNA-1 may serve useful for understanding virus-cell interactions, virus-mediated cellular transformation, and as therapies for EBV-associated pathologies.

Assay Overview:The purpose of this assay is to identify compounds that inhibit the binding of EBNA-1 protein to DNA. In this biochemical assay, recombinant EBNA-1 DNA binding domain (DBD) (aa 448-610) purified from E. coli is incubated with a Cyanine (Cy5)-labeled 36-bp hairpin DNA oligonucleotide probe in the presence of test compounds. The reaction is excited with linear polarized light and the intensity of the emitted light is measured as the polarization value (mP). As designed, compounds that act as EBNA inhibitors will prevent EBNA-DNA binding, thereby increasing the proportion of free DNA in the well, resulting in low fluorescence polarization in the well. Compounds are tested in singlicate at a final nominal concentration of 14.8 micromolar.Protocol Summary:Prior to the start of the assay, Assay Buffer (20 mM Tris HCl, pH 7.4; 200 mM NaCl; 1 mM DTT; 10 micrograms/ml BSA; 10 nM Cy5 DNA probe) containing 246 nM of EBNA-1 protein was prepared and incubated for 30 minutes at room temperature. Next, 4.0 microliters of Assay Buffer were dispensed into 1536-well microtiter plates.The assay started by dispensing 60 nL of test compound in DMSO or DMSO alone (1.5% final concentration) to the appropriate wells. Plates were then centrifuged and incubated for 1 hour at room temperature.Fluorescence polarization was read on an Envision microplate reader (PerkinElmer, Turku, Finland) using a Cy5 FP filter set (Excitation = 620nm, Emission = 688nm) and a Cy5 dichroic mirror. The well Fluorescence Polarization value (mP) was calculated from the parallel (S) and perpendicular (P) polarization values corrected with the G factor value (G) using the following formula: mP = 1000 * (S - G * P) / (S + G * P)The percent inhibition for each compound was calculated as follows:Percent inhibition = ( Test_Compound_mP -median_High_Control_mP ) / ( median_Low_Control_mP - median_High_Control_mP ) * 100Where:Test_Compound is defined as wells containing EBNA-1 in the presence of test compound.Low_Control is defined as wells containing EBNA-1.High_Control is defined as wells containing no EBNA-1.A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.The activity score range for active compounds is 100-2, for inactive 2-1.List of Reagents:Recombinant EBNA-1 (supplied by Assay Provider)Cy5 DNA probe (supplied by Assay Provider)Tris HCl (Sigma, part T1503)NaCl (Fisher, part S640-10)DTT (Sigma, part D9779)BSA (Calbiochem, part 126609)1536-well plates (Greiner, part 789176)

Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.